Thesis (PhD)--Stellenbosch University, 2012. / ENGLISH ABSTRACT: Apart from its primary function in the synaptic hydrolysis of acetylcholine, acetylcholinesterase (AChE) has been shown through in vitro demonstrations to be able to promote various non-cholinergic functions, including cell adhesion and neurite outgrowth, differentiation, and amyloidosis. AChE was also shown to bind to mouse laminin-111 in vitro by an electrostatic mechanism. Previous results suggest that the site on AChE recognised by certain monoclonal antibodies (MAbs) might be critical for differentiation. These MAbs were found to inhibit both laminin binding and cell adhesion in neuroblastoma cells. In this study, the structure and characteristics of this site were investigated, using the AChE-laminin interaction as a template as well as a detailed epitope analysis of the MAbs. The interaction sites of AChE and laminin were investigated using phage display, modelling and docking, synthetic peptides, enzyme linked immunosorbent assays (ELISAs) and conformational interaction site mapping. Docking of AChE with the single-chain variable fragments (scFvs) produced from the phage display showed the major recognition motifs to be the 90Arg-Glu-Leu-Ser-Glu-Asp motif, the 40Pro-Pro-Met-Gly sequence, and the 59Val-Val-Asp-Ala-Thr-Thr (human) motif. Mouse AChE was found to interact with the basic structures Val2718-Arg-Lys-Arg- Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg-Lys2793; and Val2817-Glu-Arg-Lys2820, on the 1 G4 domain of laminin. ELISAs using synthetic peptides confirmed the involvement of the AG-73 site (2719-2729). This site overlaps with laminin’s heparin-binding site. Docking showed the major component of the interaction site on AChE to be the acidic Arg90-Glu-Leu-Ser-Glu-Asp95 (omega loop), and also involving the Pro40-Pro-Val42, Arg46 (linked to Glu94 by a salt bridge) and the hexapeptide Asp61 Ala-Thr-Thr-Phe-Gln66. Epitope analysis showed the MAb’s major recognition site to be the sequence Pro40-Pro- Met-Gly-Pro-Arg-Arg-Phe48 (human AChE). The MAbs also reacted with the prolinerich sequences Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 and Pro88-Asn-Arg-Glu-Leu-Ser-Glu- Asp95. These results define the interaction sites involved in the AChE-laminin interaction and suggest that the interaction plays a role in cell adhesion. Despite the in vitro demonstrations of the importance of AChE’s non-classical functions, the AChE knockout survives. Results from this study suggest the possibility of functional redundancy between AChE and other molecules in early development. Using these in vitro findings that AChE is able to bind laminin-111, information on the interaction sites, as well as results from the monoclonal antibody (MAb) epitope analysis, the idea of redundancy was investigated. Docking and bioinformatics techniques were used to investigate structurally similar molecules that have comparable spatiotemporal expression patterns in the embryonic nervous system. AChE has been shown to be involved in the pathogenesis of Alzheimer’s disease, thus molecules associated with brain function and neurodegeneration were also investigated. Molecules with which AChE could be possibly redundant are syndecans, glypicans, perlecan, neuroligins and the low-density lipoprotein receptors and their variants. AChE was observed to dock with growth arrest-specific protein 6 (Gas6) as well as apolipoprotein E3 (ApoE-3) at the same site as the laminin interaction. The AChE interaction site was shown to resemble the apolipoprotein-binding site on the low density lipoprotein receptor, and related molecules, including the low density lipoprotein receptor-related molecule (LRP) and the sortilin-related receptor (SORL1). These molecules, along with apoE, are associated with Alzheimer’s disease. Resemblances to the triggering receptor on myeloid cells (TREM1) were also suggested; this is interesting as AChE has been implicated in both haematopoiesis and haematopoietic cancers. Coimmunoprecipitation results, applied to investigate alternative ligands for AChE, confirmed the AChE-laminin interaction in neuroblastoma cells, and also suggested the existence of other binding partners. In conclusion, characterisation of the AChE-laminin interaction sites and investigation of structurally similar sites in other molecules suggests a role for AChE in the stabilization of the basement membrane of developing neural cells and provides a feasible explanation for the survival of the knockout mouse. Furthermore, the demonstrated similarity of the AChE interaction site to sites on molecules, notably the low density lipoprotein receptor family and SORL1 and their apolipoprotein ligands that are implicated in the pathology of Alzheimer’s disease, as well as the possible link to haematopoietic differentiation and cancers, warrants further investigation. / AFRIKAANSE OPSOMMING: Talle in vitro studies wys dat die ensiem asetielcholienesterase (AChE), behalwe vir sy klassieke rol in die hidrolise van asetielcholien (ACh), ‘n aantal nie-cholinerge rolle vertolk insluitend in sel adhesie, in die uitgroei van neurieten, in differensiering, asook in amyloidosis. Dit is vooraf gewys dat AChE, met behulp van elektrostatiese meganismes, in vitro met muis laminin-111 kan bind. Dit word verneem dat die area op AChE wat herken word deur monoklonale teenliggaampies (MAbs), moontlik ‘n kritiese area is met betrekking tot differensiasie. Dieselfde MAbs is gevind om beide die laminin-interaksie, sowel as sel adhesie van neuroblastoma selle, te inhibeer. In hierdie projek word die struktuur en eienskappe van die betrokke kritiese areas ondersoek deur die AChE-laminin interaksie te gebruik as sjabloon. ‘n Gedetailleerde analise van die teenliggaam epitoop het ook geskied. Met behulp van faag vertoon, modellering en hegting, sintetiese peptiede, ensiem-gekoppelde immunosorbent toetse (ELISAs) en konformasie interaksie area kartering, is die betrokke interaksie areas bestudeer. Hegting van enkel-ketting varierende fragment (scFv) volgordes, verkry vanaf die vaag vertoon, aan AChE dui dat die hoof herkennings motiewe die 90Arg-Glu-Leu-Ser-Glu-Asp motief, die 40Pro-Pro- Met-Gly volgorde, en die 59Val-Val-Asp-Ala-Thr-Thr (mens) motief is. ‘n Interaksie tussen muis AChE en die 1 G4 domein van laminin is gevind. Die interaksie betrek die basiese structure: Val2718-Arg-Lys-Arg-Leu2722; Tyr2738-Tyr2739, Tyr2789-Ile-Lys-Arg- Lys2793; en Val2817-Glu-Arg-Lys2820. Die betrokkenheid van die AG-73 (2719-2729) area by hierdie interaksie is bevestig met ELISA eksperimente wat sintetiese peptiede inkorporeer. Die AG-73 area oorvleuel die heparin interaksie area op laminin. Hegtings eksperimente wys dat die hoof komponent van die interaksie area op AChE die suur volgorde Arg90-Glu-Leu-Ser-Glu-Asp95 op die omega-lus is. Die interaksie betrek ook die Pro40-Pro-Val42, Arg46 (gekoppel aan Glu94 deur ‘n sout-brug) en die heksapeptied Asp61 Ala-Thr-Thr-Phe-Gln66 motiewe. Analise van die MAb epitoop wys die hoof erkennings area as volgorde Pro40-Pro-Met-Gly-Pro-Arg-Arg-Phe48 (mens AChE). Die MAbs blyk ook gunstig te wees teenoor prolien-ryke volgordes soos Pro78-Gly-Phe-Glu-Gly-Thr-Glu84 en Pro88-Asn-Arg-Glu-Leu-Ser-Glu-Asp95. Die areas betrokke by die AChElaminin interaksie is dus gedefinieer en ‘n moontlike rol vir hierdie interaksie in sel adhesie word voorgestel. Die noodsaaklikheid van AChE se nie-klassieke funksies word bevraagteken na die oorlewing van die AChE uitklop-muis. Resultate hier dui op die moontlikheid van funksionele oortolligheid as verduideliking hiervan, spesifiek met betrekking tot molekules betrokke in vroëe ontwikkeling asook in die proses van neurale agteruitgang. Deur gebruik te maak van die in vitro demonstrasies van die AChE-laminin interaksie, informasie verkry ten opsigte van die betrokke interaksie areas, asook resultate verkry vanaf die monoklonale teenliggaam (MAb) epitoop analise, word die idee van funksionele oortolligheid ondersoek. Hegtings en bioinformatika tegnieke is gebruik om molekules met soortgelyke strukture en uitdrukkings patrone in die embrioniese senuweestelses te ondersoek. Ko-immuno presipitasie tegnieke is gebruik om so moontlike alternatiewe ligande vir AChE te ondersoek. Moontlike funksionele oortolligheid van AChE met die volgende molekules is gevind: syndecan; glypican; perlecan; neuroligin; asook die lae-digtheid lipoproteien (LDL) reseptore en hul variante. Hegting van AChE met ’growth arrest-specific’ proteien 6 (Gas6) en die apolipoproteien E3 (apoE3) is gedemonstreer en gevind om dieselfde area as die laminin interaksie te betrek. Die betrokke interaksie area op AChE het ooreenstemminge met die apolipoproteien interaksie area op die LDL reseptor asook met verwante molekules soos die lae-digtheids lipoproteien reseptor-geassosieerde molekuul (LRP) en die sortilingeassosieerde reseptor (SORL1). Hierdie molekules, insluitend apoE, speel beduidende rolle in die patologie van Alzheimer se siekte. Ooreenkomste tussen AChE en die verwekkings reseptor op myeloïde selle (TREM1) is ook voorgestel, die interaksie is van belang siende dat AChE voorheen geassosieer is met beide haematopoiesis en haematopoietiese kankers. Ko-immuno presipitasie resultate bevestig die AChE-laminin interaksie en dui op die moontlike teenwoordigheid van alternatiewe ligande vir AChE in vivo. In konklusie, karakterisering van die AChE-laminin interaksie areas, gepaard met identifisering van struktureel ooreenstemmende areas in ander molekules, dui op ‘n rol vir AChE in die stabilisering van die basale membraan en verskaf dus ‘n geldige verduideliking vir die oorlewing van die AChE uitklop-muis. Die ooreenstemming van die AChE interaksie area met areas op ander molekules (spesifiek geassosieer met Alzheimer se siekte), asook die moontlike assosiasie van AChE met haematopoietiese differensiering en kanker, lê die grondslag vir verdere ondersoeke.

Homocysteine (Hcy) has been identified as a primary risk factor for atherosclerosis as it induces endothelial cell (EC) activation/dysfunction and thus potentially initiating atherosclerotic plaque formation. There is accumulating evidence indicating a key role for oxidative stress in mediating Hcy atherogenic effects. The aim of this study was to evaluate the effects of chronic treatment with Hcy on EC activation and to explore the role of oxidative stress in these effects. Human umbilical vein endothelial cells (HUVEC) were cultured and treated chronically with DL-Hcy for 5-9 days. An in vitro flow system was also used to characterize the different types of interactions between DL-Hcy-treated HUVEC and neutrophils under physiological flow conditions. EC activation was studied by characterizing the activation of the JNK pathway and the up-regulation of different cell adhesion molecules (CAM) and cytokines, using different techniques including western blot, immunohistochemical staining, enzyme-linked immunosorbent assay and polymerase chain reaction. The role of oxidative stress was investigated by measuring the production of ROS and evaluating the efficiency of antioxidants. Furthermore, the role of nitric oxide and nitric oxide synthase in modulating Hcy effects was investigated. Chronic treatment with DL-Hcy did not kill the EC however, it inhibited cell proliferation. Furthermore, this treatment induced EC activation/dysfunction which was characterized by sustained activation of the JNK pathway, which in turn mediated up-regulation of E-selectin, ICAM-1 and to lesser extent P-selectin. Furthermore, DL-Hcy induced production of IL-8 protein. These CAM and chemokines collectively mediated different interactions between DL-Hcy-treated HUVEC and neutrophils under flow conditions including tethering, rolling, adherence and transmigration. DL-Hcy was also shown to induce significant ROS generation which mediated activation of the JNK pathway. Antioxidants restored DL-Hcy-induced interactions under flow to the basal level. DL-Hcy was shown to induce eNOS uncoupling which mediated, at least in part, the DL-Hcy-induced ROS production. Furthermore, short term treatment with NO inhibited DL-Hcy-induced HUVEC:neutrophil interactions in a cGMP-independent manner. In summary, this research showed that DL-Hcy has several proatherogenic effects, mediated at least in part by the JNK pathway, and induces EC activation/dysfunction priming for atherosclerosis initiation. The data supports that oxidative stress mediates the majority of Hcy atherosclerotic effects. Antioxidants tested, JNK inhibitors and NO showed promising results in reversing all DL-Hcy effects and restoring EC normal status.

Widefield surface plasmon resonance (WSPR) microscopy provides high resolution imaging of interfacial interactions. We report the application of the WSPR imaging system in the study of the interaction between keratinocytes and liquid crystals (LC). Imaging of fixed keratinocytes cultured on gold coated surface plasmon substrates functionalized with a thin film of liquid crystals was performed in air using a 1.45NA objective based system. Focal adhesion of the cells adhered to glass and LC were further studied using immunofluorescence staining of the vinculin. The imaging system was also simulated with 2x2 scattering matrix to investigate the optical reflection of the resonant plasmonic wave via the glass/gold/cell and glass/gold/LC/cell layers. WSPR imaging indicated that keratinocytes are less spread and formed distinct topography of cell-liquid crystal couplings when cultured on liquid crystal coated substrates. The simulation indicates that glass/LC shifted the surface plasmon excitation angle to 75.39 degrees as compared to glass/air interface at 44 degrees . The WSPR microcopy reveals that the cells remodelled their topography of adhesion at different interfaces.

Adhesion of hematopoietic cells to the bone marrow microenvironment is important for their proper development. It is proven that Src-family kinases (SFK) regulate cell adhesion, although their exact role in the regulation of adhesion signaling remains unclear. Since adhesion processes are investigated mainly in adherent cell types, far less is known about hematopoietic cells. However, defects in the cell adhesion accompany a number of hematological diseases, like chronic myeloid leukaemia (CML). SFK overexpression is one of the proposed mechanisms of resistance to the first-line CML treatment, imatinib mesylate. Second generation drugs (e. g. dasatinib) inhibit SFK together with Bcr-Abl. Additionally, SFK-specific inhibitors (PP2, Src inhibitor-1) are also available, but there are no studies about effects of these drugs on cellular adhesivity of hematopoietic precursors. To explore the dynamics of hematopoietic cell adhesion to the extracellular matrix, we introduced a new approach using the RTCA xCELLigence DP system along with the well-established method of fluorimetric detection of adherent cell fraction. Our general observation is that various drugs (dasatinib, imatinib, PP2, Src inhibitor-1) induce pro-adhesive effects in several leukemic cell lines. Direct comparison of the kinetics of...

L'activation des lymphocytes T est initié à la fois de TCR et l'engagement du co-récepteur. CD28 est le plus important sur ​​les cellules T naïves. Cette activation doit être strictement réglementé, depuis son apparition inexacte pourrait être de conséquences néfastes. Nous avons signalé que TCR et CD28 début signalisation génèrent mécanisme de détection de coïncidence dans l'initiation de l'activation des cellules T naïves. Tout d'abord, nous avons constaté que TCR déclenchement avec ligand apparenté pMHC ou anticorps augmente considérablement la liaison 2D du CD28 à ses ligands B7 et dépend à la fois la queue cytoplasmique de CD28 et l'activité de Src kinases. En outre, on a observé une interaction TCR-pMHC pour améliorer la phosphorylation sur tyrosine de CD28 induite lors de l'engagement de B7. L'analyse du récepteur déclenché par événements de signalisation dans les cellules CD4 + naïves cellules T ont montré que seul TCR ou la stimulation de CD28 est seulement capable d'induire une Ca2 faible ou minimal + réponse en dépit de la phospholipase facilement détectée C- une phosphorylation, mais la stimulation concomitante des deux voies suscité efficacement forte et soutenue + entrée Ca2 impliquant les canaux CRAC. Ainsi, notre étude a révélé apparition de la détection de coïncidence à deux étapes importantes au cours de la TCR et CD28 déclenchée par l'activation des cellules T naïves, se liant à savoir le ligand et le déclenchement des récepteurs et la mobilisation intracellulaire, qui fournit d'importantes nouvelles connaissances sur le mécanisme de l'initiation de la réponse immunitaire primaire, ainsi que sa régulation. / T cell activation is initiated by signaling pathways triggered upon ligand engagement of the TCR and co-stimulatory receptors, respectively, with CD28 being the major one among the latter class of molecules on naïve T cells. At the same time, such activation needs to be tightly regulated, since its improper occurrence might be of detrimental consequences. We report here that interactions between TCR and CD28 early signaling pathways generate coincidence detection mechanism in the initiation of naïve T cell activation. First, we found that in naïve CD4+ T cells, TCR engagement with pMHC cognate ligand or antibody significantly increases the 2D binding of CD28 to its B7 ligands and this increase depends on both the cytoplasmic tail of CD28 and activity of src kinases. Moreover, TCR-pMHC interaction was observed to enhance the tyrosine phosphorylation of CD28 induced upon B7 engagement. Analysis of the receptor-triggered signaling events in naïve CD4+ T cells showed that alone TCR or CD28 stimulation was only capable of inducing a weak or minimal Ca2+ response in spite of the readily detected phospholipase C-1 phosphorylation, but the concurrent stimulation of both pathways efficiently elicited strong and sustained Ca2+ mobilization involving the CRAC channels. Our study has thus uncovered occurrence of the coincidence detection at two major steps during the TCR- and CD28-triggered activation of naïve T cells, namely the ligand binding and triggering of the receptors and the intracellular mobilization, which provides important new insights into the mechanism of primary immune response initiation as well as its regulation.

A própolis é uma resina elaborada por abelhas que há anos tem sido utilizada na medicina popular devido as suas atividades biológica sendo que estas são atribuídas principalmente aos compostos fenólicos. Apesar disto, o mecanismo de ação da própolis permanece obscuro e a maioria dos estudos de atividade antimicrobiana realizados utilizaram métodos clássicos in vitro, que embora confirmem esta ação, não explicam como ela ocorre. Neste estudo foram avaliadas as características físico-quimicas dos extratos de própolis verde, dourada, vermelha e extrato padronizado comercial (EPP-AF®). Também foi verificada a atividade antibacteriana destes extratos contra Staphylococcus aureus ATCC 25.923, Streptococcus pneumoniae ATCC 49.619 e Klebsiella pneumoniae ATCC 10.031 utilizando metodologia de microdiluição em caldo e avaliação em cultura de células eucarióticas. Finalmente, foi avaliada a influência do extrato de própolis verde em concentrações bactericidas na morfologia das bactérias citadas. Os resultados obtidos mostraram que todos os extratos possuem grande quantidade de compostos fenólicos, entretanto a própolis vermelha apresentou melhor atividade antimicrobiana para todas as bactérias avaliadas, com concentração bactericida mínima entre 0,19 - 0,77mg/ml . Apesar disto, o extrato de própolis vermelha foi o mais instável, havendo perda significativa dos flavonoides, uma das principais substâncias pertencentes aos compostos fenólicos relacionados à atividade antimicrobiana. Além disso, verificouse que o extrato de própolis não influenciou na adesão e invasão bacteriana em células de carcinoma de laringe humano (HEp-2). As morfologias das bactérias tratadas com própolis avaliada por microscopia eletrônica de varredura evidenciou lise na superfície das bactérias gram-positivas e alteração morfológica na gramnegativa. A partir dos dados obtidos foi possível caracterizar diferentes extratos de própolis e confirmar a sua atividade antimicrobiana. Além disso, embora o mecanismo de ação não tenha sido completamente elucidado, os resultados indicam que ele está relacionado a danos estruturais provocados pela própolis e não a uma ação direta na adesão e invasão bacteriana nas células eucarióticas / Propolis is a resin produced by bees used since antiquity in folk medicine because of biological properties, especially due to phenolic compounds. However, the mechanism of action of propolis is unknown and the majority of studies on antimicrobial activity were based on in vitro classical methods that confirmed this action, but do not explain how. This present work compared the chemical composition of green, golden, red and commercial extract of propolis (EPP-AF®). Also antimicrobial activity was evaluated against Staphylococcus aureus ATCC 25923, Streptococcus pneumoniae ATCC 49619 and Klebsiella pneumoniae ATCC 10031. Microdilution method and cell culture experiments were carried out. Finally, it evaluated the influence of bactericidal concentration of green propolis on the morphology of bacteria. The results indicated there were high levels of phenolic compounds in all extracts, however red propolis presented the best antimicrobial activity against all bacteria studied, with bactericide concentration in the range of 0.19 - 0.77mg/ml. Nevertheless, red propolis extract was the most unstable, with significant loss of flavonoids, one the main substances associated with antimicrobial activity. Furthermore, the extract of propolis did not influence the adhesion and invasion of bacteria in human larynx carcinoma cell (HEp-2). The study of scanning electron microscopy of bacteria treated with propolis showed cell lysis in the grampositive and morphological changes in the gram-negative bacteria. This study contributed to characterize and confirm antimicrobial activity of different propolis extracts. Although the mechanism of action was not completely elucidated, the results indicate that the antimicrobial activity is associated with bacterial cell damage by propolis and it is not due to influence direct of propolis on bacterial adhesion and invasion of the eukaryotic cells.

INTRODUÇÃO: A interação entre leucócitos e plaquetas é fundamental para o início e a progressão da reestenose e da aterosclerose. Recentemente foi evidenciado em estudos in vitro que a integrina Mac-1 dos leucócitos se liga à glicoproteína Ibalfa (GP Ibalfa) das plaquetas e que esta interação possui uma função central na firme adesão e transmigração de leucócitos em locais de deposição de plaquetas. Entretanto, não há estudos in vivo que avaliam a importância da interação entre a integrina Mac-1 dos leucócitos e a GP Ibalfa das plaquetas (alfaMbeta2-GP Ibalfa) em modelo experimental de lesão vascular. MÉTODO: Um peptídeo denominado M2 ou anticorpo anti-M2 foi desenvolvido para bloquear a interação da integrina Mac-1 dos leucócitos com a GP Ibalfa das plaquetas, visando, deste modo, inibir a adesão de leucócitos na superfície do vaso coberta por plaquetas, a proliferação celular e a hiperplasia neointimal. Este peptídeo foi injetado e comparado com anticorpo-controle em camundongos C57B1/6J submetidos à lesão vascular da artéria femoral com corda-guia. Um dia (controle: n= 6; anti-M2: n= 6), 5 dias (controle: n= 9; anti-M2: n= 9) ou 28 dias (controle: n= 9; anti-M2: n= 9) após a lesão vascular, as artérias femorais foram retiradas para a realização de morfometria e imunohistoquímica. RESULTADOS: O bloqueio da interação alfaMbeta2-GP Ibalfa promoveu redução estatisticamente significativa de 75% do número de leucócitos na camada média no primeiro dia após a lesão vascular (controle: 7,9 ± 5,0% do total de células na camada média; versus anti-M2: 2,0 ± 1,6%; p=0,021), bem como determinou diminuição estatisticamente significativa de 42% em 5 dias (controle: 42,3 ± 12,9% do total de células na neoíntima; versus anti-M2: 24,6 ± 10,8%; p=0,047) e de 58% em 28 dias do acúmulo de leucócitos na neoíntima em desenvolvimento (controle: 7,9 ± 3,0% versus anti-M2: 3,3 ± 1,3%; p=0,012). A proliferação celular na camada média do vaso em 5 dias pós-lesão vascular apresentou redução estatisticamente significativa de 64% com o bloqueio da interação alfaMbeta2-GP Ibalfa (controle: 5,0 ± 2,9% do total de células na camada média; versus anti-M2: 1,8 ± 0,5%; p=0,043), assim como houve diminuição significativa de 47% da proliferação celular na camada íntima do vaso em 28 dias (controle: 3,8 ± 1,7% do total de células na camada íntima; versus anti-M2: 2,0 ± 1,2%; p=0,047). O bloqueio da interação alfaMbeta2-GP Ibalfa também determinou redução estatisticamente significativa de 56% do espessamento intimal em 28 dias (controle: 10.395 ± 3.549um2; versus anti-M2: 4.561 ± 4.915um2; p=0,012). CONCLUSÕES: O recrutamento de leucócitos após a lesão vascular é dependente da interação alfaMbeta2-GP Ibalfa e a neutralização desta interação inibe a proliferação celular e a formação neointimal. / INTRODUCTION: The interaction between leukocytes and platelets is fundamental for the beginning and the progression of restenosis and atherosclerosis. Recent in vitro studies have shown that the leukocyte integrin Mac-1 binds to the platelet glycoprotein (GP) Ibalfa, and this interaction plays a central role in the leukocyte firm adhesion and transmigration at sites of platelet deposition. However, there is no in vivo study evaluating the importance of the integrin Mac-1 and GP Ibalfa (alfaMbeta2-GP Ibalfa) interaction in experimental models of vascular injury. METHODS: A peptide termed M2 or anti-M2 antibody was developed to block the leukocyte Mac-1 and platelet GP Ibalfa interaction, aiming to inhibit the adhesion of leukocytes to the platelet-coated surface of vessels as well as the cellular proliferation and the neointimal hyperplasia. The peptide was injected and compared with a control-antibody in C57B1/6J mice subjected to wire-induced femoral artery injury. One day (control: n= 6; anti-M2: n= 6), 5 days (control: n= 9; anti-M2: n= 9) or 28 days (control: n= 9; anti-M2: n= 9) after vascular injury, the femoral arteries were harvested for morphometry and immunohistochemistry. RESULTS: The alfaMbeta2-GP Ibalfa interaction blockade promoted a statistically significant 75% reduction in leukocytes in the medial layer on the first day after vascular injury (control: 7.9 ± 5.0% out of the total cells in the medial layer versus anti-M2: 2.0 ± 1.6%; p=0.021), as well as determined a statistically significant 42% decrease 5 days later (control: 42.3 ± 12.9% out of the total cells in the neointima versus anti-M2: 24.6 ± 10.8%; p=0.047), and a 58% decrease in leukocyte accumulation in the developing neointima 28 days later (control: 7.9 ± 3.0% versus anti-M2: 3.3 ± 1.3%; p=0.012). The cellular proliferation in the vessel medial layer 5 days after vascular injury presented a statistically significant 64% reduction by the alfaMbeta2-GP Ibalfa interaction blockade (control: 5.0 ± 2.9% out of the total cells in the medial layer versus anti-M2: 1.8 ± 0.5%; p=0.043), and there was also a significant 47% decrease in the vessel intimal layer cellular proliferation 28 days later (control: 3.8 ± 1.7% out of the total cells in the intimal layer versus anti-M2: 2.0 ± 1.2%; p=0.047). Furthermore, the alfaMbeta2-GP Ibalfa interaction blockade determined a statistically significant 56% reduction in the intimal thickening 28 days after vascular injury (control: 10,395 ± 3,549um2 versus anti-M2: 4,561 ± 4,915um2; p=0.012). CONCLUSIONS: The leukocyte recruitment after vascular injury depends on the alfaMbeta2-GP Ibalfa interaction, and its neutralization inhibits cellular proliferation and neointimal formation.

A adesão celular está ligada à formação e disseminação de metástases, a principal causa de óbito de pacientes diagnosticados com câncer. O objetivo deste trabalho foi investigar in vitro o efeito de fotossensibilizadores na adesão celular. Foram utilizadas porfirinas comerciais (PpIX, CPpI, TSPP, TMPyP e Zn(II)TMPyP) e um fotossensibilizador sintetizado através da ligação de poli-L-lisina à protoporfirina IX (PLLPpIX). A adesão celular foi estudada por RICM, técnica que permite quantificar a área de contato entre uma célula e um substrato por binarização das imagens digitais utilizando limiares apropriados. A técnica foi padronizada e revelou dois regimes de adesão celular: um limitado e outro não limitado pela quantidade de proteína de adesão adsorvida na superfície. Neste último foi observada lise celular. Todos os fotossensibilizadores estudados foram capazes de aumentar a adesão celular na ausência de irradiação comparados ao controle sem fotossensibilizador, o que não havia sido observado nos ensaios de resistência à tripsinização normalmente utilizados para estudar o efeito de fotossensibilizadores na adesão celular. Quanto maior a anfifilicidade do fotossensibilizador, maior foi o efeito na adesão, o que é explicado pela capacidade das moléculas em se intercalarem na membrana, mudando a sua rigidez. Este aumento da adesão no escuro correlaciona com a diminuição da migração segundo ensaios de ferida. A análise do padrão de expressão de integrinas na superfície celular revela que o aumento da adesão correlaciona com o aumento na expressão de αV. Quando os fotossensibilizadores estão concentrados na região perimembranar (1 minuto de incubação) e as células são irradiadas, há um aumento da adesão em relação ao controle sem fotossensibilizador, mas uma diminuição em relação ao controle tratado com o fotossensibilizador e não irradiado, o que implica que a PDT leva a uma diminuição da adesão celular e não a um aumento como reportado na literatura. Com 3h de incubação, PLLPpIX impede a adesão celular, enquanto PpIX praticamente não muda a adesão comparado ao controle não irradiado. Esta ausência do efeito da irradiação sugere que a PpIX afeta a adesão celular principalmente devido a sua intercalação na membrana e não devido à formação de espécies reativas. Com 3h de incubação os fotossensibilizadores não se encontram na membrana e, portanto, o efeito na adesão celular é indireto e também não está relacionado à diferenças na eficiência de internalização. O comportamento observado deve ter relação com diferenças de citolocalização. Outro processo que pode alterar a adesão celular é a oxidação das proteínas do soro fetal bovino. Como observado nos estudos de fotossensibilização de células, PLLPpIX foi capaz de impedir a adesão celular, diferentemente da PpIX. A maior eficiência da PLLPpIX foi associada a presença do polímero, o qual força por questões estéricas que a interação da PLLPpIX com a albumina, o componente majoritário do soro, fique restrita à superfície da proteína, deixando o fotossensibilizador disponível para interagir com o oxigênio molecular e gerar oxigênio singlete. Assim, a funcionalização com um polímero tornou a PpIX capaz de modular a adesão celular tanto agindo dentro da célula quanto na matriz extracelular. / Cell adhesion is associated to the formation and spread of metastasis, the leading cause of death in cancer patients. The aim of this study was to investigate, in vitro, the effect of photosensitizers in cell adhesion. Five commercial porphyrins (PpIX, CPpI, TSPP, TMPyP e Zn(II)TMPyP) and Protoporphyrin IX covalently tethered to poli-L-lysine (PLLPpIX) were used. Cell adhesion was mainly studied by RICM, a technique that allows quantifying the contact area between a cell and a substrate for binarization of digital images using appropriate thresholds. The technique was standardized and disclosed two systems for cell adhesion: a system limited by the amount of adhesion proteina adsorbed on the surface and another one no limited, in which cell lysis was observed. All photosensitizers were able to enhance cell adhesion in the absence of irradiation compared to control without photosensitizer, which had not been observed in the trypsinization resistance tests usually used to study the effect of photosensitizers in cell adhesion. The greater the amphiphilicity of the photosensitizer, the greater was the effect on cell adhesion. This is explained by the ability of molecules to fit in the membrane, changing its tension. This increased adhesion correlates with the decrease in migration according to wound healing assays. Analysis of the integrin expression pattern on cell surface reveals that increased adhesion correlates with increased expression of alpha V. When photosensitizers are concentrated in the perimembranar region (1 minute of incubation) and cells are irradiated, there is an increase in adhesion when compared to control without photosensitizer, but a decrease relative to controls treated with the photosensitizer without irradiation, implying that PDT leads to a reduction of cell adhesion and not to an increase as reported in the literature. With 3h of incubation PLLPpIX prevents cell adhesion, while PpIX practically does not change the adhesion compared to dark control. This lack of effect of irradiation suggests that PpIX affects cell adhesion primarily because of its intercalation into the membrane and not due to the formation of reactive species. With 3h of incubation the photosensitizers are not on the membrane and therefore the effect on cell adhesion is indirect and it is not also related to differences in uptake efficiency. The observed behavior must be related to differences in subcellular localization arising from differences in molecular structure. Another process that can alter the cell adhesion is serum protein oxidation. As noted in the studies with cells, photosensitization of serum with PLLPpIX (but not with PpIX) was capable of preventing cell adhesion. The greater efficiency of PLLPpIX was associated with the presence of the polymer, which, by the steric hindrance, forces that interaction of PLLPpIX with albumin, the major serum component, is restricted to the protein surface, leaving the photosensitizer available to interact with molecular oxygen and generate singlet oxygen. Thus, the functionalization of a polymer has turned PpIX capable of modulating cell adhesion by acting both within and outside (in extracellular matrix) the cell

Regenerative medicine holds promises for many neurodegenerative diseases such as Traumatic Brain Injury (TBI), a disorder that occurs when a sudden trauma causes damage to the brain, leading to apoptosis or necrosis of brain neurons. More than 5 million Americans suffer from TBI as a result of inability to regenerate damaged neurons. The aim of this project was to develop a biocompatible and electrically conductive substrate to promote growth and regeneration of neurons and for our long-term goal as a probe to record intracellular and multisite signals from brain. The substrate was fabricated by pyrolyzing a polymeric precursor -SPR 220.7 at temperatures higher than 700 ºC. Human Neuroblastoma cells - SK-N-MC, SY5Y and mouse teratocarcinoma cells P-19 were found to attach and proliferate on photoresist derived carbon film. Growth and differentiation of rat pheochromocytoma cell-PC12 that serves as a model for primary neurons was demonstrated. Initial examination of cell growth and differentiation was done by observing cell shape and size, and measuring the length of neurites after the cells were differentiated by NGF. Further characterization of cells cultured on photoresist derived carbon substrate was achieved by testing mRNA genes- GADPH and Tau. Findings from this investigative work would possibly help to study new approaches to promote neuronal growth and differentiation in damaged brain regions of people with TBI or in patients with other neurodegenerative disorders, such as Alzheimer's disease in regaining memories.

Orientador: Mônica Alonso Cotta / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Física Gleb Wataghin / Made available in DSpace on 2018-09-02T02:33:05Z (GMT). No. of bitstreams: 1 Monteiro_MoniellenPires_D.pdf: 5193520 bytes, checksum: fab290a9074ae3dd188d4ebb636ff0e7 (MD5) Previous issue date: 2017 / Resumo: Nesta tese investigamos processos de adesão da Xylella fastidiosa, bactéria gram-negativa, fitopatógena, que forma biofilmes no xilema de plantas. O trabalho teve como objetivos entender alguns aspectos do processo de adesão bacteriana e do mecanismo de transporte de células e biofilmes em sistemas que simulam os vasos do xilema. Consideramos em particular as estratégias utilizadas pela X.fastidiosa para aderir à superfície, dentre elas, a secreção de substâncias poliméricas extracelulares (EPS), a presença de adesinas fimbriais (pili) e afimbriais (XadA1) e moléculas sinalizadoras de detecção de quórum (relacionadas à formação de agregados). Como ponto de partida quantificamos as propriedades elásticas do sistema composto pelo EPS e células bacterianas, em diferentes tempos de cultivo bacteriano. Para isso utilizamos medidas de espectroscopia de força e Raman confocal durante os estágios iniciais de adesão celular e formação de agregados. Mostramos que a rigidez do sistema célula/EPS diminui progressivamente com o aumento do tempo de crescimento bacteriano. Verificamos que existe uma mudança no valor de rigidez em diferentes partes da célula, região polar e corpo bacteriano; os menores valores de rigidez encontrados no polo sugerem uma resposta mecânica mais flexível nesta região, associada com o ponto de adesão inicial da célula à superfície. No estudo de adesão e mobilidade da X.fastidiosa, utilizamos dispositivos microfluídicos modificados quimicamente, via funcionalização de superfície (em microcanais Polidimetilsiloxano/vidro) e via introdução de molécula de detecção de quórum (em microcanais impressos em poliácido láctico), para tornar o ambiente mais próximo ao do xilema da planta. Foram feitas funcionalizações de superfície com uma celulose sintética, simulando a composição química dos vasos do xilema (majoritariamente composto por celulose), e com a adesina XadA1, e observamos os efeitos sobre a adesão, crescimento e mobilidade celular. Verificamos que a adesina XadA1 aumenta a densidade bacteriana se comparada às demais superficies, além de aumentar a força de adesão bacteriana à superfície. Quanto a inserção de molécula sinalizadora, observamos que a presença destas moléculas no cultivo bacteriano aumenta a densidade celular e altera a forma de pequenos agregados / Abstract: In this thesis we investigated the adhesion processes of Xylella fastidiosa, a gram-negative, phytopathogenic bacterium that forms biofilms in the xylem of plants. The objective of this work was the understanding of several aspects of the bacterial adhesion process, as well as the mechanism of cell and biofilm transport in systems that simulate xylem vessels. In particular, we considered the strategies used by X.fastidiosa to adhere to a surface; among them, the secretion of extracellular polymeric substances (EPS), the presence of fimbrial (pili) and afimbrial (XadA1) adhesins and quorum detection molecules (related to cluster formation). As a starting point we quantified the elastic properties of the system composed of EPS and bacterial cells, at different times of bacterial culture. For this purpose, we used force spectroscopy and confocal Raman measurements during the initial stages of cell adhesion and cluster formation. We have shown that stiffness decreases progressively with increasing bacterial growth time. We observed that stiffness values varied along different parts of the cell, polar region and bacterial body. The lower stiffness values found at the pole suggest a more flexible mechanical response in this region, associated with the initial adhesion point of the cell to the surface. For the investigation on X.fastidiosa adhesion and mobility, we used chemically modified microfluidic devices via surface functionalization (in Polydimethylsiloxane / glass microchannels) and via the introduction of a quorum detection molecule (in microchannels printed on polylactic acid) to make the environment more closely resemble the plant xylem. Surface functionalizations were performed with a synthetic cellulose, simulating the chemical composition of xylem vessels (mainly composed of cellulose), and the XadA1 adhesin, and we observed the effects on cell adhesion, growth and mobility. Our results showed that immobilized XadA1 increased the bacterial density when compared to other surfaces studied; furthermore, it increased the bacterial adhesion force to the surface. Regarding the addition of the signaling molecules, we observed that their presence in the bacterial culture increases cell density and changes the shape of small clusters / Doutorado / Física / Doutora em Ciências / 2010/51748-7 / 479486/2012-3 / FAPESP / CNPQ